Treatment of ischemia by administration of bone marrow (BM)-derived endothelial progenitor cells (EPC) or ex vivo expanded EPC populations has been shown to enhance vascular regeneration in animal models and in clinical trials. For cell therapy to be practical in a clinical setting, simple, efficient, GMP compliant methods for cell production are needed. Aastrom has previously developed a single-pass perfusion (SPP) bioreactor technology, the AastromReplicell TM Cell Production System (ARS), designed for automated expansion of primary human cells for clinical use. In previous studies, SPP technology has been shown to enhance function and overall production of several cell types. Based on this technology, we will develop a GMP I compliant method for the ex vivo production of substantial numbers of EPC from a small volume of BM for use in cell therapy applications. First, we will identify and optimize EPC cell culture parameters in our small-scale SPP culture system. Parameters to be examined will include starting cell population, medium serum concentration, culture duration, starting cell density and frequency of medium exchange. EPC will be enumerated and output analyzed by immunostaining and in vitro assays. Optimal culture conditions from Aim 1 will be evaluated in an in vivo model for hind limb ischemia in Aim 2. Concurrently, we will begin studies in Aim 3 to develop the EPC culture process(es) in the clinical-scale ARS. Based on small-scale results, potential revisions to the current ARS platform technology will include cell bed surface modifications and software updates to accommodate optimized medium perfusion rates. These modifications will be made with the help of in-house engineers and product specialists. Cell output from the ARS will be compared to cell output from small-scale cultures. With the successful completion of Phase I studies, a Phase II program will involve continued culture optimization and in vivo evaluations of the expanded cell products in both small-scale and clinical-scale cultures to optimize the ARS for large-scale clinical trial commercialization. Once the ARS process is established, a clinical trial will be initiated for the treatment of vascular disease. Studies will also be initiated to develop EPC gene transfer protocols within the context of the ARS platform. This work will result in the manufacture of a cell therapy kit offering automated GMP production of functional EPC cells for use in treatment of peripheral and cardiovascular diseases.